RESUMO
Infection with influenza virus induces antibodies to the viral surface glycoproteins hemagglutinin and neuraminidase, and these responses can be broadly protective. To assess the breadth and magnitude of antibody responses, we sequentially infected mice, guinea pigs and ferrets with divergent H1N1 or H3N2 subtypes of influenza virus. We measured antibody responses by ELISA of an extensive panel of recombinant glycoproteins representing the viral diversity in nature. Guinea pigs developed high titers of broadly cross-reactive antibodies; mice and ferrets exhibited narrower humoral responses. Then, we compared antibody responses after infection of humans with influenza virus H1N1 or H3N2 and found markedly broad responses and cogent evidence for 'original antigenic sin'. This work will inform the design of universal vaccines against influenza virus and can guide pandemic-preparedness efforts directed against emerging influenza viruses.
Assuntos
Anticorpos Antivirais/imunologia , Reações Cruzadas/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/imunologia , Proteínas do Envelope Viral/imunologia , Adolescente , Adulto , Fatores Etários , Animais , Análise por Conglomerados , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Furões , Cobaias , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunoglobulina G/imunologia , Vírus da Influenza A/classificação , Masculino , Camundongos , Pessoa de Meia-Idade , Neuraminidase/imunologia , Proteínas Virais/imunologia , Adulto JovemRESUMO
CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.
Assuntos
Sistemas CRISPR-Cas , Endonucleases/metabolismo , Edição de Genes , Linhagem Celular Tumoral , Endonucleases/genética , Células Hep G2 , Humanos , Estrutura Molecular , Engenharia de ProteínasRESUMO
In the early spring of 2013, Chinese health authorities reported several cases of H7N9 influenza virus infections in humans. Since then the virus has established itself at the human-animal interface in Eastern China and continues to cause several hundred infections annually. In order to characterize the antibody response to the H7N9 virus we generated several mouse monoclonal antibodies against the hemagglutinin of the A/Shanghai/1/13 (H7N9) virus. Of particular note are two monoclonal antibodies, 1B2 and 1H5, that show broad reactivity to divergent H7 hemagglutinins. Monoclonal antibody 1B2 binds to viruses of the Eurasian and North American H7 lineages and monoclonal antibody 1H5 reacts broadly to virus isolates of the Eurasian lineage. Interestingly, 1B2 shows broad hemagglutination inhibiting and neutralizing activity, while 1H5 fails to inhibit hemagglutination and demonstrates no neutralizing activity in vitro. However, both monoclonal antibodies were highly protective in an in vivo passive transfer challenge model in mice, even at low doses. Experiments using mutant antibodies that lack the ability for Fc/Fc-receptor and Fc/complement interactions suggest that the protection provided by mAb 1H5 is, at least in part, mediated by the Fc-fragment of the mAb. These findings highlight that a protective response to a pathogen may not only be due to neutralizing antibodies, but can also be the result of highly efficacious non-neutralizing antibodies not readily detected by classical in vitro neutralization or hemagglutination inhibition assays. This is of interest because H7 influenza virus vaccines induce only low hemagglutination inhibiting antibody titers while eliciting robust antibody titers as measured by ELISA. Our data suggest that these binding but non-neutralizing antibodies contribute to protection in vivo.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos Virais/imunologia , Western Blotting , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Citometria de Fluxo , Humanos , Subtipo H7N9 do Vírus da Influenza A , Camundongos , Camundongos Endogâmicos BALB CRESUMO
To evaluate the antigenic relationship between bat mumps virus (BMV) and the JL5 vaccine strain of mumps virus (MuVJL5), we rescued a chimeric virus bearing the F and HN glycoproteins of BMV in the background of a recombinant JL5 genome (rMuVJL5). Cross-reactivity and cross-neutralization between this chimeric recombinant MuV bearing the F and HN glycoproteins of BMV (rMuVJL5-F/HNBMV) virus and rMuVJL5 were demonstrated using hyperimmune mouse serum samples and a curated panel of human serum. All mouse and human serum samples that were able to neutralize rMuVJL5 infection had cross-neutralizing activity against rMuVJL5-F/HNBMV. Our data suggest that persons who have neutralizing antibodies against MuV might be protected from infection by BMV.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Quirópteros/virologia , Reações Cruzadas , Vírus da Caxumba/imunologia , Adolescente , Adulto , Animais , Feminino , Humanos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Vírus da Caxumba/isolamento & purificação , Adulto JovemRESUMO
Background: The outbreak of novel avian H7N9 influenza virus infections in China in 2013 has demonstrated the continuing threat posed by zoonotic pathogens. Deciphering the immune response during natural infection will guide future vaccine development. Methods: We assessed the induction of heterosubtypic cross-reactive antibodies induced by H7N9 infection against a large panel of recombinant hemagglutinins and neuraminidases by quantitative enzyme-linked immunosorbent assay, and novel chimeric hemagglutinin constructs were used to dissect the anti-stalk or -head humoral immune response. Results: H7N9 infection induced strong antibody responses against divergent H7 hemagglutinins. Interestingly, we also found induction of antibodies against heterosubtypic hemagglutinins from both group 1 and group 2 and a boost in heterosubtypic neutralizing activity in the absence of hemagglutination inhibitory activity. Kinetic monitoring revealed that heterosubtypic binding/neutralizing antibody responses typically appeared and peaked earlier than intrasubtypic responses, likely mediated by memory recall responses. Conclusions: Our results indicate that cross-group binding and neutralizing antibody responses primarily targeting the stalk region can be elicited after natural influenza virus infection. These data support our understanding of the breadth of the postinfection immune response that could inform the design of future, broadly protective influenza virus vaccines.
Assuntos
Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/biossíntese , Formação de Anticorpos , Especificidade de Anticorpos , China/epidemiologia , Reações Cruzadas , Surtos de Doenças , Feminino , Humanos , Influenza Humana/epidemiologia , Influenza Humana/virologia , Masculino , Pessoa de Meia-IdadeRESUMO
UNLABELLED: Between November 2013 and February 2014, China reported three human cases of H10N8 influenza virus infection in the Jiangxi province, two of which were fatal. Using hybridoma technology, we isolated a panel of H10- and N8-directed monoclonal antibodies (MAbs) and further characterized the binding reactivity of these antibodies (via enzyme-linked immunosorbent assay) to a range of purified virus and recombinant protein substrates. The H10-directed MAbs displayed functional hemagglutination inhibition (HI) and neutralization activity, and the N8-directed antibodies displayed functional neuraminidase inhibition (NI) activity against H10N8. Surprisingly, the HI-reactive H10 antibodies, as well as a previously generated, group 2 hemagglutinin (HA) stalk-reactive antibody, demonstrated NI activity against H10N8 and an H10N7 strain; this phenomenon was absent when virus was treated with detergent, suggesting the anti-HA antibodies inhibited neuraminidase enzymatic activity through steric hindrance. We tested the prophylactic efficacy of one representative H10-reactive, N8-reactive, and group 2 HA stalk-reactive antibody in vivo using a BALB/c challenge model. All three antibodies were protective at a high dose (5 mg/kg). At a low dose (0.5 mg/kg), only the anti-N8 antibody prevented weight loss. Together, these data suggest that antibody targets other than the globular head domain of the HA may be efficacious in preventing influenza virus-induced morbidity and mortality. IMPORTANCE: Avian H10N8 and H10N7 viruses have recently crossed the species barrier, causing morbidity and mortality in humans and other mammals. Although these reports are likely isolated incidents, it is possible that more cases may emerge in future winter seasons, similar to H7N9. Furthermore, regular transmission of avian influenza viruses to humans increases the risk of adaptive mutations and reassortment events, which may result in a novel virus with pandemic potential. Currently, no specific therapeutics or vaccines are available against the H10N8 influenza virus subtype. We generated a panel of H10- and N8-reactive MAbs. Although these antibodies may practically be developed into therapeutic agents, characterizing the protective potential of MAbs that have targets other than the HA globular head domain will provide insight into novel antibody-mediated mechanisms of protection and help to better understand correlates of protection for influenza A virus infection.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Imunização Passiva/métodos , Fatores Imunológicos/administração & dosagem , Vírus da Influenza A Subtipo H10N8/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Peso Corporal , Modelos Animais de Doenças , Feminino , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Fatores Imunológicos/imunologia , Pulmão/virologia , Camundongos Endogâmicos BALB C , Neuraminidase/imunologia , Testes de Neutralização , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Análise de Sobrevida , Resultado do Tratamento , Carga Viral , Proteínas Virais/imunologiaRESUMO
Three cases of influenza A(H10N8) virus infection in humans have been reported; 2 of these infected persons died. Characterization of the receptor binding pattern of H10 hemagglutinin from avian and human isolates showed that both interact weakly with human-like receptors and maintain strong affinity for avian-like receptors.
Assuntos
Hemaglutininas/fisiologia , Vírus da Influenza A Subtipo H10N8/fisiologia , Receptores Virais/fisiologia , Animais , Linhagem Celular Tumoral , Cães , Hemaglutininas/química , Humanos , Células Madin Darby de Rim Canino , Ligação Proteica , Receptores Virais/química , Ligação Viral , Replicação ViralRESUMO
UNLABELLED: Influenza virus infections are a major public health concern and cause significant morbidity and mortality worldwide. Current vaccines are effective but strain specific due to their focus on the immunodominant globular head domain of the hemagglutinin (HA). It has been hypothesized that sequential exposure of humans to hemagglutinins with divergent globular head domains but conserved stalk domains could refocus the immune response to broadly neutralizing epitopes in the stalk. Humans have preexisting immunity against H1 (group 1 hemagglutinin), and vaccination with H5 HA (also group 1)--which has a divergent globular head domain but a similar stalk domain--represents one such sequential-exposure scenario. To test this hypothesis, we used novel reagents based on chimeric hemagglutinins to screen sera from an H5N1 clinical trial for induction of stalk-specific antibodies by quantitative enzyme-linked immunosorbent assay (ELISA) and neutralization assays. Importantly, we also investigated the biological activity of these antibodies in a passive transfer in a mouse challenge model. We found that the H5N1 vaccine induced high titers of stalk-reactive antibodies which were biologically active and protective in the passive-transfer experiment. The induced response showed exceptional breadth toward divergent group 1 hemagglutinins but did not extend to group 2 hemagglutinins. These data provide evidence for the hypothesis that sequential exposure to hemagglutinins with divergent globular head domains but conserved stalk domains can refocus the immune response toward the conserved stalk domain. Furthermore, the results support the concept of a chimeric hemagglutinin universal influenza virus vaccine strategy that is based on the same principle. IMPORTANCE: Influenza virus vaccines have to be reformulated and readministered on an annual basis. The development of a universal influenza virus vaccine could abolish the need for this cumbersome and costly process and would also enhance our pandemic preparedness. This study addressed the following questions, which are essential for the development of a hemagglutinin stalk-based universal influenza virus vaccine. (i) Can stalk-reactive antibodies be boosted by vaccination with divergent HAs that share conserved epitopes? (ii) How long-lived are these vaccine-induced stalk-reactive antibody responses? (iii) What is the breadth of this reactivity? (iv) Are these antibodies functional and protective? Our results further strengthen the concept of induction of stalk-reactive antibodies by sequential exposure to hemagglutinin immunogens with conserved stalk and divergent head domains. A universal influenza virus vaccine based on the same principles seems possible and might have a significant impact on global human health.
Assuntos
Anticorpos Antivirais/sangue , Reações Cruzadas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Adulto , Animais , Anticorpos Neutralizantes/sangue , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunização Passiva , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/administração & dosagem , Masculino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Testes de Neutralização , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Adulto JovemRESUMO
The recent outbreak of H7N9 influenza virus infections in humans in China has raised concerns about the pandemic potential of this strain. Here, we test the efficacy of H3 stalk-based chimeric hemagglutinin universal influenza virus vaccine constructs to protect against H7N9 challenge in mice. Chimeric hemagglutinin constructs protected from viral challenge in the context of different administration routes as well as with a generic oil-in-water adjuvant similar to formulations licensed for use in humans.
Assuntos
Subtipo H7N9 do Vírus da Influenza A , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Adjuvantes Imunológicos , Animais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Camundongos , Infecções por Orthomyxoviridae/imunologia , Proteínas Recombinantes de Fusão/genéticaRESUMO
Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.
Assuntos
Teste para COVID-19/métodos , RNA Viral/isolamento & purificação , SARS-CoV-2/genética , Adulto , COVID-19/diagnóstico , Feminino , Humanos , Masculino , Programas de Rastreamento/métodos , RNA/genética , RNA/isolamento & purificação , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Robótica/métodos , Saliva/química , Manejo de Espécimes/métodosRESUMO
Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI-FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against the gold standard, nasopharyngeal swab specimens. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.
RESUMO
Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2-3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.
Assuntos
COVID-19/virologia , Ribonuclease P/genética , SARS-CoV-2/genética , Águas Residuárias/virologia , Primers do DNA/genética , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Vigilância Epidemiológica Baseada em Águas ResiduáriasRESUMO
Regular surveillance testing of asymptomatic individuals for SARS-CoV-2 has been center to SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay. Results of both the testing protocol itself and the study participants' experience show how the program succeeded in providing routine, robust testing capable of contributing to outbreak prevention within a campus community and offer strategies for encouraging participation and a sense of civic responsibility.
Assuntos
COVID-19/diagnóstico , Avaliação de Programas e Projetos de Saúde , Saliva/virologia , Adulto , Idoso , COVID-19/epidemiologia , COVID-19/virologia , Teste para COVID-19/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Normas Sociais , Inquéritos e Questionários , Universidades , Adulto JovemRESUMO
Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.
RESUMO
Seasonal influenza virus vaccines are generally effective at preventing disease, but need to be well matched to circulating virus strains for maximum benefit. Influenza viruses constantly undergo antigenic changes because of their high mutation rate in the immunodominant haemagglutinin (HA) head domain, which necessitates annual re-formulation and re-vaccination for continuing protection. In case of pandemic influenza virus outbreaks, new vaccines need to be produced and quickly distributed. Novel influenza virus vaccines that redirect the immune response towards more conserved epitopes located in the HA stalk domain may remove the need for annual vaccine re-formulation and could also protect against emergent pandemic strains to which the human population is immunologically naive. One approach to create such universal influenza virus vaccines is the use of constructs expressing chimeric HAs. By sequential immunization with vaccine strains expressing the same conserved HA stalk domain and exotic HA heads to which the host is naive, antibodies against the stalk can be boosted to high titres. Here we tested a monovalent chimeric HA-based prototype universal influenza virus split virion vaccine candidate with and without AS03 adjuvant in primed mice. We found that the chimeric HA-based vaccination regimen induced higher stalk antibody titres than the seasonal vaccine. The stalk antibody responses were long lasting, cross-reactive to distantly related HAs and provided protection in vivo in a serum transfer challenge model. The results of this study are promising and support further development of a universal influenza vaccine candidate built on the chimeric HA technology platform.
RESUMO
Pathogenic H7N9 avian influenza viruses continue to represent a public health concern, and several candidate vaccines are currently being developed. It is vital to assess if protective antibodies are induced following vaccination and to characterize the diversity of epitopes targeted. Here we characterized the binding and functional properties of twelve H7-reactive human antibodies induced by a candidate A/Anhui/1/2013 (H7N9) vaccine. Both neutralizing and non-neutralizing antibodies protected mice in vivo during passive transfer challenge experiments. Mapping the H7 hemagglutinin antigenic sites by generating escape mutant variants against the neutralizing antibodies identified unique epitopes on the head and stalk domains. Further, the broadly cross-reactive non-neutralizing antibodies generated in this study were protective through Fc-mediated effector cell recruitment. These findings reveal important properties of vaccine-induced antibodies and provide a better understanding of the human monoclonal antibody response to influenza in the context of vaccines.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/farmacologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Modelos Animais de Doenças , Cães , Feminino , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologiaRESUMO
Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.
Assuntos
Expressão Gênica , Genoma de Inseto , Manduca/genética , Animais , Perfilação da Expressão Gênica , Larva/genética , Larva/crescimento & desenvolvimento , Manduca/crescimento & desenvolvimento , Pupa/genética , Pupa/crescimento & desenvolvimento , Análise de Sequência de DNA , SinteniaRESUMO
Three human cases of H10N8 viruses were reported in China in late 2013 and early 2014, two of which were fatal. This was the first time the H10N8 subtype has been detected in humans and no vaccine candidates or antibody therapy has been developed for these viruses so far. We developed an H10N8 vaccine candidate virus based on A/Jiangxi-Donghu/346/13 that can also be used in a murine challenge model for vaccine and monoclonal antibody research. The vaccine virus is a 6:2 re-assortant virus expressing the surface glycoproteins of A/Jiangxi-Donghu/346/13 on an A/Puerto Rico/8/34 backbone. Vaccination with inactivated challenge virus or recombinant hemagglutinin or neuraminidase derived from this strain protected mice from viral challenge.
Assuntos
Vírus da Influenza A Subtipo H10N8/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Peso Corporal , China , Modelos Animais de Doenças , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A Subtipo H10N8/genética , Camundongos Endogâmicos BALB C , Neuraminidase/genética , Neuraminidase/imunologia , Infecções por Orthomyxoviridae/imunologia , Análise de Sobrevida , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Current influenza virus vaccines provide solid protection from infection with viruses that are well matched with the vaccine strains. However, they do not protect efficiently against drifted or shifted strains. We developed an antigen based on the conserved stalk domain of the influenza virus hemagglutinin and tested its efficacy as a vaccine in a mouse virus challenge model. Although the antigen lacked the correct conformation of the native stalk domain and was not recognized by a panel of neutralizing stalk-reactive antibodies, it did induce considerable protection against H1N1, H5N1 and H6N1 challenge strains. Protection was enhanced when mice had pre-existing immunity against the stalk domain. Since pre-existing immunity is also present in the human population, we hypothesize that a similar antigen could show efficacy in humans as well.
Assuntos
Proteção Cruzada , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Vacinação/métodos , Animais , Modelos Animais de Doenças , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae , Resultado do TratamentoRESUMO
The order of Lepidoptera has historically been crucial for chemosensory research, with many important advances coming from the analysis of species like Bombyx mori or the tobacco hornworm, Manduca sexta. Specifically M. sexta has long been a major model species in the field, especially regarding the importance of olfaction in an ecological context, mainly the interaction with its host plants. In recent years transcriptomic data has led to the discovery of members of all major chemosensory receptor families in the species, but the data was fragmentary and incomplete. Here we present the analysis of the newly available high-quality genome data for the species, supplemented by additional transcriptome data to generate a high quality reference gene set for the three major chemosensory receptor gene families, the gustatory (GR), olfactory (OR) and antennal ionotropic receptors (IR). Coupled with gene expression analysis our approach allows association of specific receptor types and behaviors, like pheromone and host detection. The dataset will provide valuable support for future analysis of these essential chemosensory modalities in this species and in Lepidoptera in general.